To improve the expression level of xylanase A in an Escherichia coli derived cell-free protein synthesis (CFPS) system, the mutation of the second codon of the signal peptide sequence (SPS) to AAA triplets was designed in xylA gene. Furthermore, the over-expression of molecular chaperons GroES-GroEL in the E. coli cell extract and the addition of Triton X-100 were also adopted to enhance the solubility and activity of the in vitro synthesized xylanase A. With the rational intrinsic manipulation and external modification, a combined strategy was established here to increase the functional expression level of xylanase A as much as 6.1-fold in CFPS. This strategy was further applied to produce other four enzymes in vitro with 3.2-fold to 5.3-fold improvements. Moreover, a modified DNA gel technique with a practical fabrication process was integrated into CFPS, resulting in a further 2.3-fold increase in the expression efficiency of xylanase A. (c) 2012 Published by Elsevier Ltd.